Respiratory syncytial virus (RSV) causes severe lower respiratory tract infection in children, especially in infants less than 1 year of age. There are currently no licensed vaccines against RSV. rA2ΔM2-2 is a promising live-attenuated vaccine candidate that is currently being evaluated in the clinic. Attenuation of rA2ΔM2-2 is achieved by a single deletion of the M2-2 gene, which disrupts the balance between viral transcription and replication. Whilst performing a manufacturing feasibility study in a serum-free adapted Vero cell line, differences in growth kinetics and cytopathic effect (CPE) were identified between two rA2ΔM2-2 vaccine candidates. Comparative sequence analysis identified four amino acid differences between the two vaccine viruses. Recombinant rA2ΔM2-2 viruses carrying each of the four amino acid differences identified a K66E mutation in the F2 fragment of the fusion (F) protein as the cause of the growth and CPE differences. Syncytium-formation experiments with RSV F protein carrying mutations at aa 66 suggested that a change in charge at this residue within the F2 fragment can have a significant impact on fusion.
The 5′ untranslated region (5′UTR) of the recently described non-primate hepacivirus (NPHV) contains a region with sequence homology to the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) and GB virus B (GBV-B). Here, we demonstrated internal translation initiation by the NPHV 5′UTR in a bicistronic vector. An RNA stem–loop upstream of the NPHV IRES was structurally distinct from corresponding regions in HCV and GBV-B, and was not required for IRES function. Insertion of the NPHV stem–loop into the corresponding region of the HCV 5′UTR within the HCV subgenomic replicon significantly impaired RNA replication, indicating that long-range interactions between the 5′UTR and cis-acting downstream elements within the NPHV genome are not interchangeable with those of HCV. Despite similarities in IRES structure and function between hepaciviruses, replication elements in the NPHV 5′UTR appear functionally distinct from those of HCV.
Picornaviruses replicate their genomes in association with cellular membranes. While enteroviruses are believed to utilize membranes of the early secretory pathway, the origin of the membranes used by foot-and-mouth disease virus (FMDV) for replication are unknown. Secretory-vesicle traffic through the early secretory pathway is mediated by the sequential acquisition of two distinct membrane coat complexes, COPII and COPI, and requires the coordinated actions of Sar1, Arf1 and Rab proteins. Sar1 is essential for generating COPII vesicles at endoplasmic reticulum (ER) exit sites (ERESs), while Arf1 and Rab1 are required for subsequent vesicle transport by COPI vesicles. In the present study, we have provided evidence that FMDV requires pre-Golgi membranes of the early secretory pathway for infection. Small interfering RNA depletion of Sar1 or expression of a dominant-negative (DN) mutant of Sar1a inhibited FMDV infection. In contrast, a dominant-active mutant of Sar1a, which allowed COPII vesicle formation but inhibited the secretory pathway by stabilizing COPII coats, caused major disruption to the ER–Golgi intermediate compartment (ERGIC) but did not inhibit infection. Treatment of cells with brefeldin A, or expression of DN mutants of Arf1 and Rab1a, disrupted the Golgi and enhanced FMDV infection. These results show that reagents that block the early secretory pathway at ERESs have an inhibitory effect on FMDV infection, while reagents that block the early secretory pathway immediately after ER exit but before the ERGIC and Golgi make infection more favourable. Together, these observations argue for a role for Sar1 in FMDV infection and that initial virus replication takes place on membranes that are formed at ERESs.
Cauliflower mosaic virus (CaMV) encodes a 520 aa polypeptide, P6, which participates in several essential activities in the virus life cycle including suppressing RNA silencing and salicylic acid-responsive defence signalling. We infected Arabidopsis with CaMV mutants containing short in-frame deletions within the P6 ORF. A deletion in the distal end of domain D-I (the N-terminal 112 aa) of P6 did not affect virus replication but compromised symptom development and curtailed the ability to restore GFP fluorescence in a GFP-silenced transgenic Arabidopsis line. A deletion in the minimum transactivator domain was defective in virus replication but retained the capacity to suppress RNA silencing locally. Symptom expression in CaMV-infected plants is apparently linked to the ability to suppress RNA silencing. When transiently co-expressed with tomato bushy stunt virus P19, an elicitor of programmed cell death in Nicotiana tabacum, WT P6 suppressed the hypersensitive response, but three mutants, two with deletions within the distal end of domain D-I and one involving the N-terminal nuclear export signal (NES), were unable to do so. Deleting the N-terminal 20 aa also abolished the suppression of pathogen-associated molecular pattern-dependent PR1a expression following agroinfiltration. However, the two other deletions in domain D-I retained this activity, evidence that the mechanisms underlying these functions are not identical. The D-I domain of P6 when expressed alone failed to suppress either cell death or PR1a expression and is therefore necessary but not sufficient for all three defence suppression activities. Consequently, concerns about the biosafety of genetically modified crops carrying truncated ORFVI sequences appear unfounded.
Herpes simplex virus type 1 establishes latency within neurons of the trigeminal ganglion. During latency, viral gene expression is largely restricted to the latency-associated transcripts (LATs), which, whilst not essential for any aspect of latency, function to suppress lytic gene expression and enhance the survival of virus-infected neurons. The latent cell population comprises primary-order neurons infected directly from peripheral tissues and cells infected following further virus spread within the ganglion. In order to assess the role of LAT expression on latency establishment within first-order neurons, we infected ROSA26R reporter mice with Cre recombinase-expressing recombinant viruses harbouring deletion of the thymidine kinase lytic gene and/or the core LAT promoter. We found that LAT expression did not impact on latency establishment in viruses unable to replicate in neurons, and under these conditions, it was not required for the survival of neurons between 3 and 31 days post-infection.
Staphylococcus aureus is one of the most important pathogens, causing various diseases in humans and animals. As methicillin-resistant S. aureus (MRSA) has become increasingly prevalent, controlling this pathogen with standard antibiotic treatment has become challenging. Bacteriophages (phages) have attracted interest as alternative antibacterial agents to control MRSA. In this study, we isolated six S. aureus phages from soils of poultry/livestock farms. Based on the results of host range determination with 150 S. aureus strains and restriction enzyme treatment of phage DNA, two phages, designated SP5 and SP6, were selected for further characterization and genome sequencing. Both SP5 and SP6 were classified as members of the family Siphoviridae. The genome of SP5 comprises 43 305 bp and contains 63 ORFs, while the SP6 genome comprises 42 902 bp and contains 61 ORFs. Although they have different host spectra, the phage genomes exhibit high nucleotide similarity to each other. Adsorption assay results suggested that the host range determinants of the two phages are involved in both adsorption and infection. Comparative genomic analyses of the two phages provided evidence that the lysogenic/lytic control module and tail proteins may be important for host specificity.
Dynamin2 is a large GTPase that regulates vesicle trafficking, and the GTPase activity of dynamin2 is required for the multistep process of adenovirus infection. Activity of dynamin2 may be regulated by post-translational phosphorylation and S-nitrosylation modifications. In this study, we demonstrate a role for dynamin2 S-nitrosylation in adenovirus infection of epithelial cells. We show that adenovirus serotype 5 (Ad5) infection augments production of nitric oxide (NO) in epithelial cells and causes the S-nitrosylation of dynamin2, mainly on cysteine 86 (C86) and 607 (C607) residues. Forced overexpression of dynamin2 bearing C86A and/or C607A mutations decreases Ad5 infection. Diminishing NO synthesis by RNAi-induced knockdown of endogenous endothelial NO synthase (eNOS) expression attenuates virus infection of target cells. Ad5 infection promotes the kinetically dynamic S-nitrosylation of dynamin2 and eNOS: there is a rapid decrease in eNOS S-nitrosylation and a concomitant increase in the dynamin2 S-nitrosylation. These results support the hypothesis that dynamin2 S-nitrosylation following eNOS activation facilitates adenovirus infection of host epithelial cells.
Hepatitis E virus (HEV) is an important but extremely understudied human pathogen. Genotypes 1 and 2 are restricted to humans, whereas genotypes 3 and 4 are zoonotic, infecting both humans and pigs. This report describes, for the first time, the successful rescue of infectious HEV in vitro and in vivo from cloned cDNA of a genotype 4 human HEV (strain TW6196E). The complete genomic sequence of the TW6196E virus was determined and a full-length cDNA clone (pHEV-4TW) was assembled. Capped RNA transcripts from the pHEV-4TW clone were replication competent in Huh7 cells and infectious in HepG2/C3A cells. Pigs inoculated intrahepatically with capped RNA transcripts from pHEV-4TW developed an active infection, as evidenced by faecal virus shedding and seroconversion, indicating the successful rescue of infectious genotype 4 HEV and cross-species infection of pigs by a genotype 4 human HEV. To demonstrate the utility of the genotype 4 HEV infectious clone and to evaluate the potential viral determinant(s) for species tropism, four intergenotypic chimeric clones were constructed by swapping various genomic regions between genotypes 1 and 4, and genotypes 1 and 3. All four chimeric clones were replication competent in Huh7 cells, but only the two chimeras with sequences swapped between genotypes 1 and 4 human HEVs produced viruses capable of infecting HepG2/C3A cells. None of the four chimeras was able to establish a robust infection in pigs. The availability of a genotype 4 HEV infectious clone affords an opportunity to delineate the molecular mechanisms of HEV cross-species infection in the future.
The PB1-F2 protein of the influenza A viruses (IAVs) can act as a virulence factor in mice. Its contribution to the virulence of IAV in swine, however, remains largely unexplored. In this study, we chose two genetically related H3N2 triple-reassortant IAVs to assess the impact of PB1-F2 in virus replication and virulence in pigs. Using reverse genetics, we disrupted the PB1-F2 ORF of A/swine/Wisconsin/14094/99 (H3N2) (Sw/99) and A/turkey/Ohio/313053/04 (H3N2) (Ty/04). Removing the PB1-F2 ORF led to increased expression of PB1-N40 in a strain-dependent manner. Ablation of the PB1-F2 ORF (or incorporation of the N66S mutation in the PB1-F2 ORF, Sw/99 N66S) affected the replication in porcine alveolar macrophages of only the Sw/99 KO (PB1-F2 knockout) and Sw/99 N66S variants. The Ty/04 KO strain showed decreased virus replication in swine respiratory explants, whereas no such effect was observed in Sw/99 KO, compared with the wild-type (WT) counterparts. In pigs, PB1-F2 did not affect virus shedding or viral load in the lungs for any of these strains. Upon necropsy, PB1-F2 had no effect on the lung pathology caused by Sw/99 variants. Interestingly, the Ty/04 KO-infected pigs showed significantly increased lung pathology at 3 days post-infection compared with pigs infected with the Ty/04 WT strain. In addition, the pulmonary levels of interleukin (IL)-6, IL-8 and gamma interferon were regulated differentially by the expression of PB1-F2. Taken together, these results indicate that PB1-F2 modulates virus replication, virulence and innate immune responses in pigs in a strain-dependent fashion.
Members of the family Picornaviridae are important pathogens of humans and animals, although compared with the thousands of known bird species (>10 000), only a few (n = 11) picornaviruses have been identified from avian sources. This study reports the metagenomic detection and complete genome characterization of a novel turkey picornavirus from faecal samples collected from eight turkey farms in Hungary. Using RT-PCR, both healthy (two of three) and affected (seven of eight) commercial turkeys with enteric and/or stunting syndrome were shown to be shedding viruses in seven (88 %) of the eight farms. The viral genome sequence (turkey/M176/2011/HUN; GenBank accession no. JQ691613) shows a high degree of amino acid sequence identity (96 %) to the partial P3 genome region of a picornavirus reported recently in turkey and chickens from the USA and probably belongs to the same species. In the P1 and P2 regions, turkey/M176/2011/HUN is related most closely to, but distinct from, the kobuviruses and turdivirus 1. Complete genome analysis revealed the presence of characteristic picornaviral amino acid motifs, a potential type II-like 5′ UTR internal ribosome entry site (first identified among avian-origin picornaviruses) and a conserved, 48 nt long ‘barbell-like’ structure found at the 3′ UTR of turkey/M176/2011/HUN and members of the picornavirus genera Avihepatovirus and Kobuvirus. The general presence of turkey picornavirus – a novel picornavirus species – in faecal samples from healthy and affected turkeys in Hungary and in the USA suggests the worldwide occurrence and endemic circulation of this virus in turkey farms. Further studies are needed to investigate the aetiological role and pathogenic potential of this picornavirus in food animals.
To determine the extent to which influenza viruses jump between human and swine hosts, we undertook a large-scale phylogenetic analysis of pandemic A/H1N1/09 (H1N1pdm09) influenza virus genome sequence data. From this, we identified at least 49 human-to-swine transmission events that occurred globally during 2009–2011, thereby highlighting the ability of the H1N1pdm09 virus to transmit repeatedly from humans to swine, even following adaptive evolution in humans. Similarly, we identified at least 23 separate introductions of human seasonal (non-pandemic) H1 and H3 influenza viruses into swine globally since 1990. Overall, these results reveal the frequency with which swine are exposed to human influenza viruses, indicate that humans make a substantial contribution to the genetic diversity of influenza viruses in swine, and emphasize the need to improve biosecurity measures at the human–swine interface, including influenza vaccination of swine workers.
Dengue virus (DENV) causes dengue fever and dengue haemorrhagic fever/dengue shock syndrome, both considered major public-health problems worldwide. We generated a lethal DENV-2 strain (D220) by 10 additional cycles of subcutaneous inoculation of mice with supernatant from mosquito cells infected with the previously characterized strain D2S10, followed by harvesting of serum. D220 induces mortality at ten-fold lower doses than D2S10 in mice lacking only the alpha/beta interferon (IFN-α/β) receptor in C57BL/6 or 129 backgrounds under both non-enhanced and antibody-enhanced conditions. Sequence analysis of the complete viral genome revealed five amino acid changes between D220 and D2S10, of which two (K122I in envelope and V115A in NS4B) appear to account for the observed phenotypic differences between the viruses. By causing mortality at lower doses in C57BL/6 mice lacking only the IFN-α/β receptor, D220 constitutes an improved tool for study of DENV-induced pathogenesis, as well as for testing potential vaccines and antiviral drugs against DENV.
Thirteen novel avipoxviruses were isolated from birds from different regions of South Africa. These viruses could be divided into six groups, according to gross pathology and pock appearance on chick chorioallantoic membranes (CAMs). Histopathology revealed distinct differences in epidermal and mesodermal cell proliferation, as well as immune cell infiltration, caused by the different avipoxviruses, even within groups of viruses causing similar CAM gross pathology. In order to determine the genetic relationships among the viruses, several conserved poxvirus genetic regions, corresponding to vaccinia virus (VACV) A3L (fpv167 locus, VACV P4b), G8R (fpv126 locus, VLTF-1), H3L (fpv140 locus, VACV H3L) and A11R–A12L (fpv175–176 locus) were analysed phylogenetically. The South African avipoxvirus isolates in this study all grouped in clade A, in either subclade A2 or A3 of the genus Avipoxvirus and differ from the commercial fowlpox vaccines (subclade A1) in use in the South African poultry industry. Analysis of different loci resulted in different branching patterns. There was no correlation between gross morphology, histopathology, pock morphology and phylogenetic grouping. There was also no correlation between geographical distribution and virus phenotype or genotype.
Recent studies suggest that low levels of reactive oxygen species (ROS) often modulate normal intracellular signalling pathways, determine cell fates and control cell proliferation. We found that infection of astrocytes with the neuropathogenic retrovirus ts1, a mutant of Moloney murine leukemia retrovirus, upregulated ROS at low levels during the early phase of infection. This upregulation of intracellular ROS with downregulation of NADPH levels during the early phase of ts1 infection was a separate event from the upregulation of ROS during the late phase while ts1-mediated cell death occurred. The treatment of apocynin, a potential inhibitor of NADPH oxidase (NOX), inhibited establishment of the ts1 virus in the host cell. These results suggested that ROS generated as a consequence of the activation of NOX may play an important role in the early events of the virus life cycle leading to the establishment of the virus in the host cell. The in vitro results were further supported by an in vivo experiment which showed that the treatment of apocynin decreased viral titre in the ts1-infected mouse brain and increased the lifespan of infected mice. This study provides the first in vitro and in vivo evidence on a mechanism for how ROS are involved in ts1 retrovirus infection and ts1-mediated neurodegenerative disease. Our findings focusing on the early phase of the ts1 retrovirus life cycle could provide a better understanding of retroviral life cycle, which may offer specific therapeutic targets for suppressing viral replication and alleviating neurodegenerative symptoms in a mouse model.
A novel genetic approach for the control of virus replication was used for the design of a conditionally replicating human immunodeficiency virus (HIV) variant, HIV-rtTA. HIV-rtTA gene expression and virus replication are strictly dependent on the presence of a non-toxic effector molecule, doxycycline (dox), and thus can be turned on and off at will in a graded and reversible manner. The in vivo replication capacity, pathogenicity and genetic stability of this HIV-rtTA variant were evaluated in a humanized mouse model of haematopoiesis that harbours lymphoid and myeloid components of the human immune system (HIS). Infection of dox-fed BALB Rag/γc HIS (BRG-HIS) mice with HIV-rtTA led to the establishment of a productive infection without CD4+ T-cell depletion. The virus did not show any sign of escape from dox control for up to 10 weeks after the onset of infection. No reversion towards a functional Tat–transactivating responsive (TAR) RNA element axis was observed, confirming the genetic stability of the HIV-rtTA variant in vivo. These results demonstrate the proof of concept that HIV-rtTA replicates efficiently in vivo. HIV-rtTA is a promising tool for fundamental research to study virus–host interactions in vivo in a controlled fashion.
Each year, influenza viruses cause epidemics by evading pre-existing humoral immunity through mutations in the major glycoproteins: the haemagglutinin (HA) and the neuraminidase (NA). In 2004, the antigenic evolution of HA of human influenza A (H3N2) viruses was mapped (Smith et al., Science
305, 371–376, 2004) from its introduction in humans in 1968 until 2003. The current study focused on the genetic evolution of NA and compared it with HA using the dataset of Smith and colleagues, updated to the epidemic of the 2009/2010 season. Phylogenetic trees and genetic maps were constructed to visualize the genetic evolution of NA and HA. The results revealed multiple reassortment events over the years. Overall rates of evolutionary change were lower for NA than for HA1 at the nucleotide level. Selection pressures were estimated, revealing an abundance of negatively selected sites and sparse positively selected sites. The differences found between the evolution of NA and HA1 warrant further analysis of the evolution of NA at the phenotypic level, as has been done previously for HA.
We have developed a porcine intestine epithelial cell line, designated SD-PJEC for the propagation of influenza viruses. The SD-PJEC cell line is a subclone of the IPEC-J2 cell line, which was originally derived from newborn piglet jejunum. Our results demonstrate that SD-PJEC is a cell line of epithelial origin that preferentially expresses receptors of oligosaccharides with Sia2-6Gal modification. This cell line is permissive to infection with human and swine influenza A viruses and some avian influenza viruses, but poorly support the growth of human-origin influenza B viruses. Propagation of swine-origin influenza viruses in these cells results in a rapid growth rate within the first 24 h post-infection and the titres ranged from 4 to 8 log10 TCID50 ml−1. The SD-PJEC cell line was further tested as a potential alternative cell line to Madin–Darby canine kidney (MDCK) cells in conjunction with 293T cells for rescue of swine-origin influenza viruses using the reverse genetics system. The recombinant viruses A/swine/North Carolina/18161/02 (H1N1) and A/swine/Texas/4199-2/98 (H3N2) were rescued with virus titres of 7 and 8.25 log10 TCID50 ml−1, respectively. The availability of this swine-specific cell line represents a more relevant substrate for studies and growth of swine-origin influenza viruses.
Human enterovirus 68 (EV-D68) is a historically rarely reported virus linked with respiratory disease. In the past 3 years, a large increase in respiratory disease associated with EV-D68 has been reported, with documented outbreaks in North America, Europe and Asia. In several outbreaks, genetic differences were identified among the circulating strains, indicating the presence of multiple clades. In this report, we analyse archived and novel EV-D68 strains from Africa and the USA, obtained from patients with respiratory illness. Phylogenetic analysis of all EV-D68 sequences indicates that, over the past two decades, multiple clades of the virus have emerged and spread rapidly worldwide. All clades appear to be currently circulating and contributing to respiratory disease.
Vaccinia virus (VACV) expresses many proteins that are non-essential for virus replication but promote virulence by inhibiting components of the host immune response to infection. These immunomodulators include a family of proteins that have, or are predicted to have, a structure related to the B-cell lymphoma (Bcl)-2 protein. Five members of the VACV Bcl-2 family (N1, B14, A52, F1 and K7) have had their crystal structure solved, others have been characterized and a function assigned (C6, A46), and others are predicted to be Bcl-2 proteins but are uncharacterized hitherto (N2, B22, C1). Data presented here show that N2 is a nuclear protein that is expressed early during infection and inhibits the activation of interferon regulatory factor (IRF)3. Consistent with its nuclear localization, N2 inhibits IRF3 downstream of the TANK-binding kinase (TBK)-1 and after IRF3 translocation into the nucleus. A mutant VACV strain Western Reserve lacking the N2L gene (vΔN2) showed normal replication and spread in cultured cells compared to wild-type parental (vN2) and revertant (vN2-rev) viruses, but was attenuated in two murine models of infection. After intranasal infection, the vΔN2 mutant induced lower weight loss and signs of illness, and virus was cleared more rapidly from the infected tissue. In the intradermal model of infection, vΔN2 induced smaller lesions that were resolved more rapidly. In summary, the N2 protein is an intracellular virulence factor that inhibits IRF3 activity in the nucleus.
Prior to the identification of hepatitis C virus (HCV), transfusion-transmission was common. Viral transmission in subjects with a known date of infection allows the study of the immune responses to acute HCV infection. We analysed 39 soluble immune factors in serum samples from subjects with transfusion-transmitted HCV. Dynamic expression kinetics of interferon gamma-induced protein 10 (IP-10), tumour necrosis factor-alpha and interleukin (IL)-10 were observed during acute HCV infection. Serum IP-10 was the only analyte that was significantly elevated in HCV resolvers compared with uninfected controls. In individuals who progressed to chronic HCV elevated levels of IP-10 and IL-10 coincided with first significant alanine aminotransferase elevation and remained elevated during the first year of acute HCV infection. In addition to monitoring lack of reduction in viral load, serum levels of IP-10 and IL-10 expression during acute HCV infection may be useful biomarkers to predict the progress to chronic HCV.
The first confirmed outbreak of highly pathogenic avian influenza (HPAI) virus infections in North America was caused by A/turkey/Ontario/7732/1966 (H5N9); however, the phylogeny of this virus is largely unknown. This study performed genomic sequence analysis of 11 avian influenza isolates from 1956 to 1979 for comparison with A/turkey/Ontario/7732/1966 (H5N9). Phylogenetic and genetic analyses included these viruses in combination with all known full-genome sequences of avian viruses isolated before 1981. It was shown that a low-pathogenic avian influenza virus, A/turkey/Ontario/6213/1966 (H5N1), that had been isolated 3 months previously, was the closest known genetic relative with six genome segments of common lineage encoding the polymerase subunits PB2, PB1 and PA, nucleoprotein (NP), haemagglutinin (HA) and non-structural (NS) proteins. The lineages of these genome segments included reassortment with other North American turkey viruses that were all rooted in North American wild waterfowl with the HA gene originating from the H5N2 serotype. The phylogenies demonstrated adaptation from North American wild birds to turkeys with the possible involvement of domestic waterfowl. The turkey isolate, A/turkey/Wisconsin/1968 (H5N9), was the second most closely related poultry isolate to A/turkey/Ontario/7732/1966 (H5N9), possessing five common lineage genome segments (PB2, PB1, PA, HA and neuraminidase). The A/turkey/Ontario/6213/1966 (H5N1) virus was more virulent than A/turkey/Wisconsin/68 (H5N9) for chicken embryos and mice, indicating a greater biological similarity to A/turkey/Ontario/7732/1966 (H5N9). Thus, A/turkey/Ontario/6213/1966 (H5N1) was identified as the closest known ancestral relative of HPAI A/turkey/Ontario/7732/1966 (H5N9), which will serve as a useful reference virus for characterizing the early genetic and biological properties associated with the emergence of pathogenic avian influenza strains.
Hepatitis E virus (HEV) is an important human pathogen. In addition to humans, HEV has also been identified in pig, chicken, mongoose, deer, rat, rabbit and fish. There are four recognized and two putative genotypes of mammalian HEV. Genotypes 1 and 2 are restricted to humans, while genotypes 3 and 4 are zoonotic. The recently identified rabbit HEV is a distant member of genotype 3. Here, we first expressed and purified the recombinant capsid protein of rabbit HEV and showed that the capsid protein of rabbit HEV cross-reacted with antibodies raised against avian, rat, swine and human HEV. Conversely, we showed that antibodies against rabbit HEV cross-reacted with capsid proteins derived from chicken, rat, swine and human HEV. Since pigs are the natural host of genotype 3 HEV, we then determined if rabbit HEV infects pigs. Twenty pigs were divided into five groups of four each and intravenously inoculated with PBS, US rabbit HEV, Chinese rabbit HEV, US rat HEV and swine HEV, respectively. Results showed that only half of the pigs inoculated with rabbit HEV had low levels of viraemia and faecal virus shedding, indicative of active but not robust HEV infection. Infection of pigs by rabbit HEV was further verified by transmission of the virus recovered from pig faeces to naïve rabbits. Pigs inoculated with rat HEV showed no evidence of infection. Preliminary results suggest that rabbit HEV is antigenically related to other HEV strains and infects pigs and that rat HEV failed to infect pigs.
Recent studies indicate that human papillomaviruses (HPVs) from the genera Betapapillomavirus and Gammapapillomavirus are abundant in the human oral cavity. We report the cloning and characterization of a 7304 bp HPV120 genome from the oral cavity that is related most closely to HPV23 (L1 ORF, 83.7 % similarity), clustering it in the genus Betapapillomavirus (β-PV). HPV120 contains five early and two late genes, but no E5 ORF. HPV120 was detected from heterogeneous human biological niches, including the oral cavity, eyebrow hairs, anal canal and penile, vulvar and perianal warts. Characterization of the clinical spectrum of HPV120 infections indicates a broader spectrum of epithelial tropism than appreciated previously for HPV types from the genus β-PV.
Syncytin-1 and envPb1 are two conserved envelope genes in the human genome encoded by single loci from the HERV-W and -Pb families, respectively. To characterize the role of these envelope proteins in cell–cell fusion, we have developed lentiviral vectors that express short hairpin RNAs for stable knockdown of syncytin-1 and envPb1. Analysis of heterotypic fusion activity between trophoblast-derived choriocarcinoma BeWo cells, in which syncytin-1 and envPb1 are specifically silenced, and endothelial cells demonstrated that both syncytin-1 and envPb1 are important to fusion. The ability to fuse cells makes syncytin-1 and envPb1 attractive candidate molecules in therapy against cancer. Our available vectors may help eventually to decipher roles for these genes in human health and/or disease.
Autographa californica multiple nucleopolyhedrovirus requires nuclear actin for progeny virus production and thereby encodes viral products that ensure actin’s translocation to and retention within the nucleus. Current evidence suggests that the ie0–ie1 gene complex along with five nuclear localization of actin (NLA) genes are sufficient for NLA in transient transfection experiments. Here we report that, during infection, only one of the five NLA genes, Ac102, was essential for NLA, and that AC102 had at least one other activity critical for budded virus (BV) production. Viral deletion mutants in the other four NLA genes were viable, with only two having replication phenotypes different from that of the wild type. Infection with AcΔpe38 revealed a delay in both BV production and NLA. Infection with AcΔ152 revealed a delay in BV production, but no corresponding delay in NLA. Infection with either AcΔpe38 or AcΔ152 resulted in slightly reduced BV titres. Deletion of Ac004 or he65 had no impact on actin translocation kinetics, timing of BV production or BV titres. These results implicate AC102 as a key player in baculovirus manipulation of actin.